Delivery of cell-killing doses of ionizing radiation to tumors is the objective of antibody-directed radioimmunotherapy (RIT). In practice, however, collateral damage to healthy bone marrow and kidneys limits the maximum delivered radiation dose. Pretargeted RIT (PRIT) aims to overcome this limitation by separating the pharmacokinetics of tumor targeting and radionuclide delivery. First-generation PRIT utilizes the streptavidin/biotin interaction for radionuclide capture, and exhibits promise in animal tumor xenograft models. However, problems with streptavidin immunogenicity, kidney localization, and endogenous biotin intrinsically limit this approach. The perspective of this proposal is that the principle of PRIT is sound, but that to reach its full potential the protein targeting agents must be optimized, and the pharmacokinetics of tumor penetration must be subjected to rigorous engineering analysis. This project brings together faculty from Biological Engineering and Radiation Oncology to collaboratively develop the essential reagents and dosing strategies to enable PRIT to be maximally effective. Protein engineering by directed evolution will be applied to: 1) construct human antibody fragments that capture radiometal chelates (DOTA with yttrium, gallium, or bismuth) effectively irreversibly;and 2) to develop anti-CEA antibody fragments that bind persistently through cycles of endocytic trafficking through acidic compartments. The processes of diffusion, binding, and metabolism of antibody fragments in micrometastases will be characterized by quantitative fluorescence microscopy of single LS174T human colon adenocarcinoma tumor cells and spheroid cultures, and analyzed within a mathematical modeling framework to determine the limiting kinetic processes and predict necessary concentrations and times for saturation binding. These predictions will be tested with quantitative biodistribution studies in LS174T xenografts in nude mice. Taken together, these studies will establish a firm foundation from which to optimize PRIT. In this second and final allowed revision of the proposal, we have eliminated radiation dosimetry and focused on endocytic trafficking of antibody fragments as a critical process limiting the permeation and retention of bispecific antibodies, based on direct evidence for rapid antibody uptake by LS174T cells. This hypothesis informs the planned efforts in protein engineering, cell culture, and in vivo biodistribution studies.